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[[ | ==Ternary complex of Bacillus DNA Polymerase I Large Fragment, DNA duplex, and dCTP (paired with dG of template)== | ||
<StructureSection load='4dqi' size='340' side='right' caption='[[4dqi]], [[Resolution|resolution]] 1.69Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[4dqi]] is a 6 chain structure with sequence from [http://en.wikipedia.org/wiki/Geobacillus_kaustophilus Geobacillus kaustophilus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4DQI OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4DQI FirstGlance]. <br> | |||
</td></tr><tr><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=DCP:2-DEOXYCYTIDINE-5-TRIPHOSPHATE'>DCP</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene><br> | |||
<tr><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=DOC:2,3-DIDEOXYCYTIDINE-5-MONOPHOSPHATE'>DOC</scene></td></tr> | |||
<tr><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[4dqq|4dqq]], [[4dqr|4dqr]], [[4dqs|4dqs]], [[4ds4|4ds4]], [[4ds5|4ds5]], [[4dse|4dse]], [[4dsf|4dsf]], [[4dqp|4dqp]]</td></tr> | |||
<tr><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">GK2730, GYMC61_0787, polA ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=1462 Geobacillus kaustophilus])</td></tr> | |||
<tr><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/DNA-directed_DNA_polymerase DNA-directed DNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.7 2.7.7.7] </span></td></tr> | |||
<tr><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4dqi FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4dqi OCA], [http://www.rcsb.org/pdb/explore.do?structureId=4dqi RCSB], [http://www.ebi.ac.uk/pdbsum/4dqi PDBsum]</span></td></tr> | |||
<table> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
In addition to discriminating against base-pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxy nucleotides. It has been proposed that a single active site residue (steric gate) blocks productive binding of nucleotides containing 2' hydroxyls. Although this steric gate plays a role in sugar moiety discrimination, its interactions do not account fully for the observed behavior of mutants. Here we present ten high-resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment (BF) variants complexed with deoxy-, ribo-, dideoxy-nucleotides, and a DNA substrate. Taken together, these data present a more nuanced and general mechanism for nucleotide discrimination in which ensembles of intermediate conformations in the active site trap non-cognate substrates. It is known that the active site O-helix transitions from an open state in the absence of nucleotide substrates to a ternary complex closed state in which the reactive groups are aligned for catalysis. Substrate misalignment in the closed state plays a fundamental part in preventing non-cognate nucleotide misincorpation. The structures presented here show that additional O-helix conformations intermediate between the open and closed state extremes create an ensemble of binding sites that trap and misalign non-cognate nucleotides. Water-mediated interactions, absent in the fully closed state, play an important role in formation of these binding sites, and can be remodeled to accommodate different non-cognate substrates. This mechanism may extend also to base-pair discrimination. | |||
Structural factors that determine selectivity of a high-fidelity DNA polymerase for deoxy-, dideoxy-, and ribo-nucleotides.,Wang W, Wu EY, Hellinga HW, Beese LS J Biol Chem. 2012 May 30. PMID:22648417<ref>PMID:22648417</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
==See Also== | |||
*[[DNA polymerase|DNA polymerase]] | |||
== | == References == | ||
[[ | <references/> | ||
__TOC__ | |||
== | </StructureSection> | ||
< | |||
[[Category: DNA-directed DNA polymerase]] | [[Category: DNA-directed DNA polymerase]] | ||
[[Category: Geobacillus kaustophilus]] | [[Category: Geobacillus kaustophilus]] | ||
Revision as of 09:56, 25 June 2014
Ternary complex of Bacillus DNA Polymerase I Large Fragment, DNA duplex, and dCTP (paired with dG of template)Ternary complex of Bacillus DNA Polymerase I Large Fragment, DNA duplex, and dCTP (paired with dG of template)
Structural highlights
Publication Abstract from PubMedIn addition to discriminating against base-pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxy nucleotides. It has been proposed that a single active site residue (steric gate) blocks productive binding of nucleotides containing 2' hydroxyls. Although this steric gate plays a role in sugar moiety discrimination, its interactions do not account fully for the observed behavior of mutants. Here we present ten high-resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment (BF) variants complexed with deoxy-, ribo-, dideoxy-nucleotides, and a DNA substrate. Taken together, these data present a more nuanced and general mechanism for nucleotide discrimination in which ensembles of intermediate conformations in the active site trap non-cognate substrates. It is known that the active site O-helix transitions from an open state in the absence of nucleotide substrates to a ternary complex closed state in which the reactive groups are aligned for catalysis. Substrate misalignment in the closed state plays a fundamental part in preventing non-cognate nucleotide misincorpation. The structures presented here show that additional O-helix conformations intermediate between the open and closed state extremes create an ensemble of binding sites that trap and misalign non-cognate nucleotides. Water-mediated interactions, absent in the fully closed state, play an important role in formation of these binding sites, and can be remodeled to accommodate different non-cognate substrates. This mechanism may extend also to base-pair discrimination. Structural factors that determine selectivity of a high-fidelity DNA polymerase for deoxy-, dideoxy-, and ribo-nucleotides.,Wang W, Wu EY, Hellinga HW, Beese LS J Biol Chem. 2012 May 30. PMID:22648417[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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